Prevalence, Incidence, and Risk Factors for Intestinal Colonization Due to Fluoroquinolone-Resistant ST131 Escherichia coli: a Longitudinal Study in Highly Dependent, Long-Term Care Facility Residents

ABSTRACT Escherichia coli ST131 clade C is an important driver for fluoroquinolone resistance (FQ-R). We conducted a prospective observational study in residents from two long-term care facilities (LTCFs) in Seville, Spain, in 2018. Fecal swabs and environmental samples were obtained. E. coli isolates were screened for clade C, FQ-R ST131 by PCR, and molecular typing by PFGE; representatives from pulsotypes were studied by whole-genome-sequencing (WGS) and assigned to lineages (cgSTs). Prevalence of colonization at each time point, incidence density, and risk factors for acquisition were studied. Seventy-six FQ-R ST131 E. coli isolates belonging to 34 cgSTs were obtained; 24 belonging to subclade C1 (116 isolates, 65.9%) and 10 to C2 (60, 34.1%). C1 lineages showed lower virulence scores than C2 (median [IQR], 19 [18 to 20] versus 21 [20 to 21.5], P = 0.001) and higher number of plasmids (4 [3 to 5] versus 2 [2 to 3], P = 0.01). aac(6’)-Ib-cr and blaOXA-1 were less frequent in C1 than C2 (2 [8.3%] versus 6 [60%], P = 0.003 for both); ESBL genes were detected in eight (33.3%) C1 (5 blaCTX-M-27) and three (30%) C2 (all blaCTX-M-15). Of the 82 residents studied, 49 were colonized at some point (59.7%), with a pooled prevalence of 38.6%. Incidence density of new lineage acquisition was 2.22 per 100 resident weeks (1.28 and 0.93 C1 and C2 subclades, respectively). Independent risk factors for acquisitions were having a colonized roommate (HR = 4.21; 95% CI = 1.71 to 10.36; P = 0.002) and urinary or fecal incontinence (HR = 2.82; 95% CI = 1.21 to 6.56; P = 0.01). LTCFs are important reservoirs of clade C ST131 E. coli. The risk factors found suggest that cross-transmission is the most relevant transmission mechanisms. IMPORTANCE We aimed at investigating the microbiological and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli isolates colonizing highly dependent residents in long-term care facilities (LTCFs) during 40 weeks and the risk factors of acquisition. Isolates from C1 and C2 subclades were characterized in this environment. The clonality of the isolates was characterized and they were assigned to lineages (cgSTs), Resistance genes, virulence factors, and plasmids were also described. This study suggests that cross-transmission is the most relevant transmission mechanisms; however, environmental colonization might also play a role. We believe the data provide useful information to depict the epidemiology of these bacteria by merging detailed microbiological and epidemiological information.

in my opinion would improve the manuscript. a)This is just a question. Why authors only focused on clade C since there are some papers showing the increasing of clade A importance. Clade A ST131 are poorly studied but in terms of lethality are considered equivalent to clade C in a mouse sepsis model of infection. Furthermore, clade A isolates also carry blaCTX-M-27 and dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a feature previously considered unique to clade C isolates. In my opinion, authors missed out here a great opportunity to enrich the work b)Page 09: Change 49 residents to 34. The sum does not match c)Page 09: "regarding virulence genes, afaA, afaC and afa D were less frequent....." Please write the complete name before the acronyms are used for the first time. The same for nfaE gene. Why these virulence factors are so important in ST131 Escherichia coli strains? This information is not contextualised in the text. d)Page 11: There is no Table 04. "The 34 residents who acquired any new strain of FQ-R ST131 E. coli were compared with the 42 who did not (table 4)" e)table 2 shows several virulence factor genes. However, in any place in the main text the authors discuss the relevance of these genes in the ability of fluoroquinolone-resistant ST131 E. coli in colonize and spread. Looks that this information was not so explored. The same for Plasmid incompatibility groups. f)In my opinion, supplementary figure should be included in the main text Reviewer #2 (Public repository details (Required)): The WGS data should be deposited in a database.
Reviewer #2 (Comments for the Author): In the manuscript Spectrum01673-22, the authors traced fluoroquinolone-resistant Escherichia coli ST131 clade C in two long term care facilities. They report that cross-transmission among individuals residing in proximity is more important as a risk factor than microbiological or other virulence factors. The work is interesting, important, and described in detail. The authors also mention the limitations of their data which must be commended. I have following comments. Major Comments: 1. The authors must emphasize how their work is different in terms of design and interpretations than their cited reference # 14 and 15. 2. The manuscript is largely descriptive. Addition of a diagram which shows incidence and transmission tree with color codes would definitely improve the manuscript. Please see Figure Table 4' in 'Risk factors for acquisition of colonization' section. There is no Table 4 in the manuscript. I suggest that the authors replace this supplementary figure with a transmission tree as a main figure. 3. Since the major claim of the study is cross-transmission through proximity, the authors should also further explain the causal link between transmission isolates and environmental isolates. The do so in the first paragraph of page 11 but, addition of a phylogenetic tree amongst all the isolates will add clarity (they already have the WGS data). Since the number of samples is on the lower side, addition of this data will propel the correlation to causation. 4. In table 3, the author should explain why the correlation with the renal disease is not taken into account. Perhaps, is it because of the n (and hence the confidence) being small? 5. The authors should expand the discussion on the virulence factors they found (table 3).
Minor Comments: 1. The authors should add line numbers in the manuscript. 2. In the abstract, please expand the LTCF abbreviation for the readers. 3. Fix the grammar in line: "was avoided because some residents transitory leave the LTCF." 4. Where were the environmental samples collected from? The authors mention toilets, shower heads, etc. but they should mention the location such as which facility and same or different rooms etc.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
The manuscript entitled "Prevalence, incidence and risk factors for intestinal colonization due to fluoroquinolone-resistant ST131 Escherichia coli: a longitudinal study in highly dependent, long term-care facility residents." concerns a prospective observational study in residents from long term-care facilities in order to investigate the colonization and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli. As known the ST131 is the most frequently Thank you for providing us the opportunity to submit a revised version of the manuscript entitled "Prevalence, incidence and risk factors for intestinal colonization due to fluoroquinolone-resistant ST131 Escherichia coli: a longitudinal study in highly-dependent, long term-care facility residents."; we also thank the reviewers for their comments which have been really helpful in improving the manuscript.
Please see our answers to your and the reviewers' comments below.
Sincerely, Jesus Rodriguez-Baño On behalf of all authors ____________________________________________________________________________ Reviewers' comments:

Reviewer #1
The manuscript entitled "Prevalence, incidence and risk factors for intestinal colonization due to fluoroquinolone-resistant ST131 Escherichia coli: a longitudinal study in highly dependent, long term-care facility residents." concerns a prospective observational study in residents from long term-care facilities in order to investigate the colonization and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli. As known the ST131 is the most frequently isolated fluoroquinolone-resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Overall, the work is well performed and results would be of interest for the journal's readership. However, there are minor few points that needed to be addressed which in my opinion would improve the manuscript. a)This is just a question. Why authors only focused on clade C since there are some papers showing the increasing of clade A importance. Clade A ST131 are poorly studied but in terms of lethality are considered equivalent to clade C in a mouse sepsis model of infection. Furthermore, clade A isolates also carry blaCTX-M-27 and dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a feature previously considered unique to clade C isolates. In my opinion, authors missed out here a great opportunity to enrich the work Response: We thank the reviewer for the general comment on our manuscript. Regarding the decision to investigate clade A, we agree with the reviewer about the interest of this clade due to lack of data; however, when the study was designed, we were particularly interested in FQ-resistant clade C and the emergence of CTX-M-27 within. b) Page 09: Change 49 residents to 34. The sum does not match Response: We are sorry but we think the data is correct. Please note there were 34 different cgSTs among all participants in which an isolate was found at any point in the study (49 residents were positive at some point in the study but some cgSTs recurred among them). c) Page 09: "regarding virulence genes, afaA, afaC and afa D were less frequent....." Please write the complete name before the acronyms are used for the first time. The same for nfaE gene. Why these virulence factors are so important in ST131 Escherichia coli strains? This information is not contextualised in the text.
Response: Thank you very much for your suggestion. We guess the reviewer refers to the name of the proteins codified by the genes. We have consulted other manuscripts published by ASM and only the names of the genes are provided. However, we will follow any indication of the Editor in this regard. We added some sentences in the Discussion to explain the interest of the findings regarding the virulence factor genes, and added two references. d) Page 11: There is no Table 04. "The 34 residents who acquired any new strain of FQ-R ST131 E. coli were compared with the 42 who did not (table 4)" Response: Thank you for raising this mistake. We corrected it. e) table 2 shows several virulence factor genes. However, in any place in the main text the authors discuss the relevance of these genes in the ability of fluoroquinolone-resistant ST131 E. coli in colonize and spread. Looks that this information was not so explored. The same for Plasmid incompatibility groups.
Response: As explained above, we added some sentences in this regard. We have looked at associations between virulence factors genes or plasmids and high transmissibility, but as explained we could not find any association., as stated already in the Discussion section. We also added the possibility that this may be due to lack of statistical power f) In my opinion, supplementary figure should be included in the main text Response: Following the reviewer suggestion, we have included the figure in the main text as Figure 1.

Reviewer #2
The WGS data should be deposited in a database.
Response: Of course, the reviewer is right. We have deposited the WGS in a database and added in the manuscript: Raw sequencing reads of 64 E. coli ST131 isolates (those obtained from patients and environment) were deposited in BioProject PRJNA742861.
In the manuscript Spectrum01673-22, the authors traced fluoroquinolone-resistant Escherichia coli ST131 clade C in two long term care facilities. They report that cross-transmission among individuals residing in proximity is more important as a risk factor than microbiological or other virulence factors. The work is interesting, important, and described in detail. The authors also mention the limitations of their data which must be commended. I have following comments.
Major Comments: 1. The authors must emphasize how their work is different in terms of design and interpretations than their cited reference # 14 and 15.
Response: Thank you for raising this important aspect. We added a sentence in the Discussion; our study provided longitudinal data allowing an assessment of transmission events while references 14, 15 and 16, while very interesting, provided point prevalence data.
2. The manuscript is largely descriptive. Addition of a diagram which shows incidence and transmission tree with color codes would definitely improve the manuscript. Please see Figure  Response: Thank you for the suggestion. Due to the high number of participants, samples and clones, over a long study period, we have considered different ways to represent the transmission events together with duration of colonization. In fact, we had considered using a similar figure to those suggested by the reviewer but we think these (the first refers to transmission of Ebola and the second mostly to viruses) are not the most appropriate for the information we needed to provide. In fact, these figures don´t capture some essential aspects such as the duration of colonization and the time elapsed among transmission events. However, if the Editor suggests us otherwise we would provide a different figure.
Indeed, there is an error when referencing that Table/ Figure, we corrected it, and as suggested, we added it as a figure within the main document. We are sorry for this mistake.
3. Since the major claim of the study is cross-transmission through proximity, the authors should also further explain the causal link between transmission isolates and environmental isolates. The do so in the first paragraph of page 11 but, addition of a phylogenetic tree amongst all the isolates will add clarity (they already have the WGS data). Since the number of samples is on the lower side, addition of this data will propel the correlation to causation.
Response: We agree with the reviewer that providing a phylogenetic tree of all isolates would be of interest. However, due to funds limitation and as explained in the text, we could perform WGS only for specific representatives in each pulsotype plus and, in addition, for those with different susceptibility phenotypes within each pulsotype. We added a sentence with this limitation in the Discussion section.
4. In table 3, the author should explain why the correlation with the renal disease is not taken into account. Perhaps, is it because of the n (and hence the confidence) being small? Re: Spectrum01673-22R1 (Prevalence, incidence and risk factors for intestinal colonization due to fluoroquinolone-resistant ST131 Escherichia coli: a longitudinal study in highly-dependent, long term-care facility residents.) Dear Prof. Jesús Rodríguez-Baño: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey.
As an open-access publication, Spectrum receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted. You will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.